The subject of this investigation is the regulation of cholesterol metabolism. Various approaches have been used to study the possible mechanisms involved in its regulation. One particular question attempted is whether feedback inhibition exerted by dietary cholesterol on 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of the sterol biosynthetic pathway, can be mediated in a cell-free system that is capable of sterol synthesis. Secondly, using antiserum prepared against purified HMG-CoA reductase and radioisotope incorporation into enzyme protein, we intend to analyze the mechanisms of inhibition of the enzyme upon short-term and long-term cholesterol feeding. In addition, isolated rat intestinal villi and crypt cells will be prepared and maintained in a culture medium to study the effects of added cholesterol or its oxygenated analogs and whether they can alter HMG-CoA reductase activity or acetate incorporation into digitonin-precipitable sterols. Another aspect of this study is to continue the investigation on enzyme-lipid interactions. Present data suggest that the enzyme possibly interacts with membrane lipids by an association with the head group moieties of phospholipids. This possibility will be tested by reconstituting enzyme into phospholipid dispersions containing electron paramagnetic resonance probes attached to phospholipid head groups.